marine drugs

Article Neuroprotective Effect of Carotenoid-Rich Enteromorpha prolifera Extract via TrkB/Akt Pathway against Oxidative Stress in Hippocampal Neuronal Cells

Seung Yeon Baek and Mee Ree Kim * Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-42-821-6837; Fax: +82-42-821-8887



Received: 27 June 2020; Accepted: 17 July 2020; Published: 19 July 2020


Abstract: In this study, we found that E. prolifera extract (EAEP) exhibits neuroprotective effects in oxidative stress-induced neuronal cells. EAEP improved cell viability as well as attenuated the formation of intracellular reactive oxygen species (ROS) and apoptotic bodies in glutamate-treated hippocampal neuronal cells (HT-22). Furthermore, EAEP improved the expression of brain-derived neurotrophic factor (BDNF) and antioxidant enzymes such as heme oxygenase-1 (HO-1), NAD(P)H quinine oxidoreductase-1 (NQO-1), and glutamate–cysteine ligase catalytic subunit (GCLC) via the tropomyosin-related kinase receptor B/ protein kinase B (TrkB/Akt) signaling pathway. In contrast, the pre-incubation of K252a, a TrkB inhibitor, or MK-2206, an Akt-selective inhibitor, ameliorated the neuroprotective effects of EAEP in oxidative stress-induced neuronal cells. These results suggest that EAEP protects neuronal cells against oxidative stress-induced apoptosis by upregulating the expression of BDNF and antioxidant enzymes via the activation of the TrkB/Akt pathway. In conclusion, such an effect of EAEP, which is rich in carotenoid-derived compounds, may justify its application as a food supplement in the prevention and treatment of neurodegenerative disorders.

Keywords: Enteromorpha prolifera; oxidative stress; apoptosis; BDNF; TrkB/Akt pathway

1. Introduction In recent decades, the number of patients diagnosed with neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, has increased continuously along with the extended lifespan and environmental pollution worldwide [1,2]. The pathophysiology of neurodegenerative diseases is closely related to the generation of reactive oxygen species (ROS), which results in protein and DNA damage, inflammation, tissue damage and subsequently induces apoptosis in neuronal cells [3,4]. Accordingly, one of the key neuroprotective strategies entails regulation of ROS generation in order to prevent or treat neurodegenerative diseases [5]. Another preventive and treatment strategy for neurodegenerative disease involves activating the production of brain-derived neurotrophic factor (BDNF) [6]. BDNF is released from the central nervous system and plays an important role in cell proliferation, protection, synaptic function, morphogenesis, and plasticity mediated via tropomyosin-related kinase receptor B (TrkB) [7,8]. Most studies investigating BDNF have reported that the expression of BDNF protects neuronal cells against oxidative stress and decreases the risk of neurodegenerative disorders in the brain [8,9]. Recent studies suggest that some natural antioxidant compounds suppress the oxidative stress induced by ROS and activate the BDNF/TrkB pathway in neuronal cells [9–11]. Marine seaweeds possess biochemical properties that may be exploited to develop therapeutic and functional foods. Marine seaweeds are considered an abundant source of antioxidants including flavonoids, phenolic compounds, carotenoids, and chlorophyll [12–15]. Enteromorpha prolifera (EP)

Mar. Drugs 2020, 18, 372; doi:10.3390/md18070372 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, 372 2 of 12

Marine seaweeds possess biochemical properties that may be exploited to develop therapeutic Mar. Drugs 2020, 18, 372 2 of 13 and functional foods. Marine seaweeds are considered an abundant source of antioxidants including flavonoids, phenolic compounds, carotenoids, and chlorophyll [12–15]. Enteromorpha prolifera (EP) belongs to the phylum phylum Chlorophyt Chlorophyta,a, class class Chlorophyceae, Chlorophyceae, order order UlvalesUlvales,, and and genus genus EnteromorphaEnteromorpha [16].[16]. It is is cultivated cultivated worldwide worldwide along along the the seashore seashore and and is used is used to treat to treat symptoms symptoms including including epistaxis epistaxis and signsand signs of inflammation of inflammation in eastern in eastern Asia Asia [17]. [17 ].Poly Polysaccharidessaccharides derived derived from from EP EP have have attracted attracted the attention of investigators involved in their identific identificationation and determination of structure and function including antioxidant,antioxidant, anti-inflammatory, anti-inflammatory, anti-diabetic, anti-diabetic, and anti-apoptoticand anti-apoptotic activities activities [17–20]. However,[17–20]. However,EP is a source EP is of a polysaccharides,source of polysaccharides, crude fiber crude and fib protein,er and asprotein, well as as unsaturated well as unsaturated fatty acids fatty such acids as suchlinoleic as linoleic acid, linolenic acid, linolenic acid, EPA, acid, DHA, EPA, minerals, DHA, mine andrals, vitamins and vitamins [21]. Furthermore, [21]. Furthermore, EP contains EP contains diverse phytochemicalsdiverse phytochemicals such as chlorophyll,such as chlorophyll, phycocyanin, phycocyanin, flavonoids, flavonoids, and phenolic and compounds, phenolic compounds, which affect whichthe antioxidant affect the activity antioxidant of the activity marine of algal the extract marine [22 algal,23]. extract The anti-diabetic [22,23]. The eff anti-diabeticect of flavonoids effect and of polyphenolsflavonoids and in thepolyphenols EP extract wasin the established EP extract in was the intestinalestablished microflora in the intestinal of type 2 microflora diabetic mice of [type24,25 2]. diabeticIn addition, mice in [24,25]. a previous In addition, report, we in investigated a previous thatreport, the antioxidantwe investigated activity that of the an ethylantioxidant acetate fractionactivity of an EP ethyl extract acetate (EAEP) fraction was stronger of EP extract than that(EAEP) of aqueous was stronger and 95%than ethanol that of aqueous extract [23 and]. In95% the ethanol report, weextract found [23]. that In the EAEP report, is rich we infound carotenoid-derived that EAEP is rich dihydroactinidiolide in carotenoid-derived and dihydroactinidiolide carotenoids including and carotenoidscanthaxanthin, including violaxanthin, canthaxant and fucoxanthin,hin, violaxanthin, which exhibit and potent fucoxa neuroprotectiventhin, which activity exhibit [23 ,potent26–29]. neuroprotectiveDespite extensive activity analyses [23,26–29]. of the EPDespite extract, extensiv few studiese analyses have of investigatedthe EP extract, the few antioxidant studies have and investigatedneuroprotective the eantioxidantffects associated and neuroprotectiv with phytochemicals.e effects associated with phytochemicals. In this study, we investigated the neuroprotectiv neuroprotectivee role of EAEP against oxidative stress-induced neurodegeneration in hippocampal neuronal cellscells (HT-22)(HT-22) mediatedmediated viavia TrkBTrkB/Akt/Aktpathway. pathway.

2. Results Results

2.1. Neuroprotective Neuroprotective Effect Effect of EAEP on Glutamate-InducedGlutamate-Induced Oxidative Stress in HT-22HT-22 CellsCells Glutamate isis an an excitatory excitatory neurotransmitter neurotransmitter in the centralin the nervous central system nervous [30]. Highsystem concentrations [30]. High concentrationsof glutamate inhibit of glutamate the uptake inhibit of Nthe-acetyl uptake cysteine, of N-acetyl which cysteine, results which in the results reduction in the in intracellularreduction in intracellularglutathione levelsglutathione and subsequent levels and subsequent oxidative stress oxidative associated stress withassociated cell death with suchcell death as apoptosis such as apoptosisand necrosis and [ 31necrosis]. First, [31]. we investigatedFirst, we invest whetherigated EAEPwhether prevented EAEP prev glutamate-inducedented glutamate-induced oxidative oxidativestress in hippocampal stress in hippocampal neuronal cellsneuronal because cells EAEP because has beenEAEP reported has been to reported exhibit high to exhibit antioxidant high antioxidantactivity [21,23 activity]. The incubation[21,23]. The of HT-22incuba cellstion withof HT-22 EAEP (10–100cells withµg /mL)EAEP prior (10–100 to glutamate μg/mL) treatment prior to glutamateimproved celltreatment viability improved up to a cell concentration viability up of to 100 a µconcentrationg/mL (Figure of?? 100A). Inμg/mL particular, (Figure exposure 1A). In particular,to EAEP 100 exposureµg/mL to completely EAEP 100 μ restoredg/mL completely cell viability restored to the cell same viability level to as the in same the controllevel as group.in the Ascontrol shown group. in Figure As shown??B, in glutamate-induced Figure 1B, glutamate- HT-22induced cells generatedHT-22 cells intracellular generated intracellular ROS. In contrast, ROS. InEAEP contrast, dose-dependently EAEP dose-dependently reduced the reduced intracellular the in ROStracellular level (Figure ROS level??B). (Figure These results 1B). These suggest results that suggestEAEP attenuated that EAEP neuronal attenuated cell deathneuronal against celloxidative death against stress byoxidative regulating stress ROS by generation. regulating Thus,ROS generation.EAEP may playThus, a EAEP neuroprotective may play a role neuroprotective in glutamate-induced role in glutamate-induced HT-22 cells. HT-22 cells.

Figure 1. Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on glutamate-induced Figure 1. Effect of ethyl acetate extract of Enteromorpha prolifera (EAEP) on glutamate-induced cytotoxicity and reduction in reactive oxygen species (ROS) in hippocampal neuronal cells (HT-22). cytotoxicity and reduction in reactive oxygen species (ROS) in hippocampal neuronal cells (HT-22). HT-22 cells, seeded on 96-well-plates and incubated for 24 h, were treated with or without EAEP HT-22 cells, seeded on 96-well-plates and incubated for 24 h, were treated with or without EAEP (0– (0–100 µg/mL) for 30 min before glutamate challenge (5 mM). After 12 h, cell viability and intracellular 100 μg/mL) for 30 min before glutamate challenge (5 mM). After 12 h, cell viability and intracellular ROS levels were estimated as described in the Materials and Methods. (A) Cell viability. (B) ROS ROS levels were estimated as described in the Materials and Methods. (A) Cell viability. (B) ROS level. level. Data represent the mean SEM values in triplicate; ** p < 0.01 versus glutamate-treated group. ± denotes absence of EAEP. − Mar. Drugs 2020, 18, 372 3 of 12 Mar. Drugs 2020, 18, 372 3 of 13 Data represent the mean ± SEM values in triplicate; ** p < 0.01 versus glutamate-treated group. − denotes absence of EAEP. 2.2. Inhibitory Effect of EAEP on Oxidative Stress-Induced Apoptosis in HT-22 Cells 2.2. Inhibitory Effect of EAEP on Oxidative Stress-Induced Apoptosis in HT-22 Cells Next, we investigated inhibitory effect of EAEP against oxidative stress in glutamate-induced HT-22 cells becauseNext, glutamate we investigated induces inhibitory apoptosis effect in of HT-22 EAEP cells against [30 ].oxidative As shown stress in in Figure glutamate-induced2, flow cytometry HT-22 cells because glutamate induces apoptosis in HT-22 cells [30]. As shown in Figure 2, flow using Annexin V led to a significant increase in apoptotic bodies in glutamate-treated HT-22 cells. cytometry using Annexin V led to a significant increase in apoptotic bodies in glutamate-treated HT- Meanwhile,22 cells. Meanwhile, pretreatment pretreatment with EAEP with dose-dependently EAEP dose-dependently reduced reduced thenumber the number of apoptoticof apoptotic bodies. Furthermore,bodies. Furthermore, treatment treatment with EAEP with 100 EAEPµg /100mL μ restoredg/mL restored the level the level of apoptotic of apoptotic bodies bodies to to that that of the controlof the group, control indicating group, indicating that EAEP that protected EAEP protec neuronalted neuronal cells bycells inhibiting by inhibiting oxidative oxidative stress-induced stress- apoptoticinduced cell apoptotic death. cell death.

FigureFigure 2. 2.Inhibitory Inhibitory eeffectffect of of ethyl ethyl acetate acetate extract extract of Enteromorpha of Enteromorpha prolifera (EAEP) prolifera on(EAEP) oxidative on stress- oxidative stress-inducedinduced apoptosis apoptosis in HT-22 in HT-22 cells. cells.HT-22 HT-22 cells, seed cells,ed seededon 60-mm on dishes 60-mm and dishes incubated and incubatedfor 24 h, were for 24 h, weretreated treated with with or without or without EAEP EAEP (0–100 (0–100 μg/mL)µ forg/mL) 30 min for before 30 min glutam beforeate challenge glutamate (5 mM). challenge After (512 mM). Afterh, 12 the h, harvested the harvested cells cellsincluding including apoptotic apoptotic and necr andotic necrotic cells were cells analyzed were analyzed by flow bycytometry flow cytometry as as describeddescribed inin SectionSection 2.2.( (AA) vehicle) vehicle control; control; (B) glutamate (B) glutamate alone; alone; (C) glutamate (C) glutamate + 10 μg/mL+ 10 EAEP;µg/mL (D) EAEP; glutamate + 25μg/mL EAEP; (E) glutamate + 50 μg/mL EAEP; (F) glutamate + 100 μg/mL EAEP. − (D) glutamate + 25µg/mL EAEP; (E) glutamate + 50 µg/mL EAEP; (F) glutamate + 100 µg/mL EAEP. denotes absence of EAEP. denotes absence of EAEP. − 2.3. EAEP2.3. EAEP Activates Activates the the Expression Expression of of Antioxidant Antioxidant Enzymes Enzymes via via Akt/Nrf2 Akt/Nrf2 Pathway Pathway We evaluated the effects of EAEP on the expression of antioxidant enzymes to identify the We evaluated the effects of EAEP on the expression of antioxidant enzymes to identify the mechanism underlying prevention of oxidative stress-induced apoptosis (Figure 3). It was reported mechanismthat activation underlying of Akt/Nrf2 prevention pathway of influenced oxidative th stress-inducede neuroprotective apoptosis activity [31,32]. (Figure Treatment3). It was with reported thatEAEP activation led to of upregulation Akt/Nrf2 pathway of the expression influenced of thep-Akt neuroprotective and the nuclear activity translocation [31,32 ].Nrf2. Treatment It also with EAEPboosted led to upregulationthe levels of ofantioxidant the expression enzymes of p-Aktinclud anding theHO-1, nuclear glutamate–cysteine translocation Nrf2.ligase Itcatalytic also boosted the levelssubunit of (GCLC), antioxidant and NQO-1 enzymes in glutamate-treated including HO-1, HT-2 glutamate–cysteine2 cells. Based on these ligase results, catalytic the subunitantioxidant (GCLC), and NQO-1action of inEAEP glutamate-treated seems to be mediated HT-22 via cells. Akt/Nrf2 Based signaling on these pathway. results, the antioxidant action of EAEP seems to be mediated via Akt/Nrf2 signaling pathway. 2.4. Effect of Activated EAEP on the Expression of BDNF Mediated via ERK/CREB/TrkB Pathway The ERK/TrkB/CREB pathway is another mechanism underlying neuronal cell survival [33,34]. As shown in Figure 4, EAEP completely enhanced the expression of BDNF as well as p-ERK, p-CREB, and p-TrkB (Figure 4A). Based on these results, the neuroprotective effect of EAEP is closely associated with the synthesis of BDNF via ERK/CREB/TrkB pathway in neuronal cells. Since EAEP protects neuronal cell against oxidative stress via the ERK/CREB/TrkB pathway, we were interested in the intermediate protein related to mitogen-activated protein kinase (MAPK). Additionally, we confirmed the expression of JNK and p38, which belong to MAPK, in oxidative stress-induced

Mar. Drugs 2020, 18, 372 4 of 12 neuronal cells. In glutamate-treated HT-22 cells, the expression of activated JNK and p38 was increased (Figure 4B). In contrast, EAEP significantly diminished their phosphorylation induced by glutamate, which was less than the control level. Therefore, we concluded that EAEP upregulated Mar. Drugs 2020, 18, 372 4 of 13 the activation of BDNF/ERK/CREB/TrkB signaling pathway and downregulated the expression of JNK and p38 in oxidative stress-induced neuronal cells, simultaneously.

FigureFigure 3. 3. TreatmentTreatment with with ethyl ethyl acetate acetate extract extract of of EnteromorphaEnteromorpha prolifera prolifera (EAEP)(EAEP) activates activates the the expression expression ofof antioxidant enzymesenzymes viavia Akt Akt/Nrf2/Nrf2 pathway. pathway. (A ()A Immunoblotting) Immunoblotting for for protein protein expression expression related related with withAkt/ Nrf2Akt/Nrf2 pathway, pathway, (B) relative (B) relative density density of p-Akt, of p-Akt, nuclear nuclear and cytosolicand cytosolic Nrf2, Nrf2, (C) NQO-1,(C) NQO-1, GLCL, GLCL, and andHO-1. HO-1. HT-22 HT-22 cells werecells seededwere seeded on a 60-mm on a 60-mm dish, and dish, incubated and incubated for 24 h. Thefor 24 cells h. wereThe challengedcells were challengedwith glutamate with glutamate after pre-incubation after pre-in withcubation or without with or without EAEP (0–100 EAEP µ(0–100g/mg) μ forg/mg) 30 min.for 30 Aftermin. After 12 h, β 12the h, expression the expression of p-Akt, of p-Akt, nuclear nuclear and cytosolic and cytosolic Nrf2, HO-1, Nrf2, NQO-1, HO-1, GCLCNQO-1, or GCLC-actin or was β-actin examined was examinedas described as indescribed the Materials in the and Materials Methods. and The Meth dataods. were The based data onwere three based independent on three independent experiments. ** p < 0.01 versus glutamate-treated group. denotes absence of EAEP. experiments. ** p < 0.01 versus glutamate-treated− group. − denotes absence of EAEP. 2.4. Effect of Activated EAEP on the Expression of BDNF Mediated via ERK/CREB/TrkB Pathway The ERK/TrkB/CREB pathway is another mechanism underlying neuronal cell survival [33,34]. As shown in Figure4, EAEP completely enhanced the expression of BDNF as well as p-ERK, p-CREB, and p-TrkB (Figure4A). Based on these results, the neuroprotective e ffect of EAEP is closely associated with the synthesis of BDNF via ERK/CREB/TrkB pathway in neuronal cells. Since EAEP protects neuronal cell against oxidative stress via the ERK/CREB/TrkB pathway, we were interested in the intermediate protein related to mitogen-activated protein kinase (MAPK). Additionally, we confirmed the expression of JNK and p38, which belong to MAPK, in oxidative stress-induced neuronal cells. In glutamate-treated HT-22 cells, the expression of activated JNK and p38 was increased (Figure4B). In contrast, EAEP significantly diminished their phosphorylation induced by glutamate, which was less than the control level. Therefore, we concluded that EAEP upregulated the activation of BDNF/ERK/CREB/TrkB signaling pathway and downregulated the expression of JNK and p38 in oxidative stress-induced neuronal cells, simultaneously.

Figure 4. Activation of brain-derived neurotrophic factor (BDNF) expression via ERK/CREB/TrkB pathway following treatment with ethyl acetate extract of Enteromorpha prolifera (EAEP). (A) Immunoblotting and relative density of BDNF, p-CREB, p-TrkB, (B) p-ERK, p-JNK, and p-p38. HT-22 cells were seeded on a 60-mm dish, and then incubated for 24 h. The cells were challenged with glutamate after pre-incubation with or without EAEP (0–100 μg/mg) for 30 min. After 12 h, the expression of BDNF, p-CREB, p-TrkB, p-ERK, p-JNK, p-38 or β-actin was examined as described in the Materials and Methods. The data were pooled from three independent experiments. ** p < 0.01 versus glutamate-treated group. − denotes absence of EAEP.

Mar. Drugs 2020, 18, 372 4 of 12 neuronal cells. In glutamate-treated HT-22 cells, the expression of activated JNK and p38 was increased (Figure 4B). In contrast, EAEP significantly diminished their phosphorylation induced by glutamate, which was less than the control level. Therefore, we concluded that EAEP upregulated the activation of BDNF/ERK/CREB/TrkB signaling pathway and downregulated the expression of JNK and p38 in oxidative stress-induced neuronal cells, simultaneously.

Figure 3. Treatment with ethyl acetate extract of Enteromorpha prolifera (EAEP) activates the expression of antioxidant enzymes via Akt/Nrf2 pathway. (A) Immunoblotting for protein expression related with Akt/Nrf2 pathway, (B) relative density of p-Akt, nuclear and cytosolic Nrf2, (C) NQO-1, GLCL, and HO-1. HT-22 cells were seeded on a 60-mm dish, and incubated for 24 h. The cells were challenged with glutamate after pre-incubation with or without EAEP (0–100 μg/mg) for 30 min. After 12 h, the expression of p-Akt, nuclear and cytosolic Nrf2, HO-1, NQO-1, GCLC or β-actin was

Mar. Drugsexamined2020, 18as, 372described in the Materials and Methods. The data were based on three independent5 of 13 experiments. ** p < 0.01 versus glutamate-treated group. − denotes absence of EAEP.

FigureFigure 4. ActivationActivation of of brain-derived brain-derived neurotrophic neurotrophic fa factorctor (BDNF) (BDNF) expression expression via via ERK/CREB/TrkB ERK/CREB/TrkB pathwaypathway following following treatment treatment with with ethyl ethyl acetate acetate extract extract of Enteromorpha of Enteromorpha prolifera prolifera (EAEP).(EAEP). (A) Immunoblotting(A) Immunoblotting and relative and relative density density of BDNF, of BDNF, p-CREB, p-CREB, p-TrkB, p-TrkB, (B) p-ERK, (B) p-ERK, p-JNK, p-JNK,and p-p38. and HT-22 p-p38. cellsHT-22 were cells seeded were seededon a 60-mm on a 60-mm dish, and dish, then and incu thenbated incubated for 24 forh. The 24 h. cells The were cells challenged were challenged with glutamatewith glutamate after pre-incubation after pre-incubation with or with without or without EAEP EAEP (0–100 (0–100 μg/mg)µg /mg)for 30 for min. 30 min.After After12 h, 12the h, β expressionthe expression of BDNF, of BDNF, p-CREB, p-CREB, p-TrkB, p-TrkB, p-ERK, p-ERK, p-JNK, p-JNK, p-38 p-38 or or β-actin-actin was was examined examined as as described described in in thethe Materials Materials and and Methods. Methods. The The data data were were p pooledooled from three independent experiments. ** ** pp < 0.010.01 versus glutamate-treated group. denotes absence of EAEP. versus glutamate-treated group. −− denotes absence of EAEP. 2.5. K252a and MK2206 Inhibit the Neuroprotective Effects of EAEP Subsequently, we found that the neuroprotective effects of EAEP affect both the BDNF/TrkB pathway and the Akt/Nrf2/antioxidant enzymes by treating with K252a, a TrkB inhibitor, or MK2206, a selective Akt inhibitor, We determined that the inhibitory effect dramatically neutralized the antioxidant and neuroprotective effects of EAEP in oxidative stress-induced HT-22 cells (Figure5). Pre-treatment with either K252a or MK2206 in combination with EAEP significantly reduced the neuroprotective effect of EAEP against glutamate-treated HT-22 cells (Figure5A,C). In addition, the inhibitor treatment interfered with the ability of EAEP to reverse ROS synthesis in oxidative stress-induced hippocampal neuronal cells (Figure5B,D). Furthermore, both K252a and MK2006 reversed the antioxidant action of EAEP by reducing the expression of antioxidant enzymes including HO-1, NQO-1, and GCLC in oxidative stress-induced HT-22 cells (Figure6). In particular, the expression of BDNF and p-TrkB was decreased when the inhibitors were pre-treated with EAEP (Figure7). Collectively, these results suggest that the activation of Akt is closely associated with the BDNF/TrkB pathway as well as the expression of antioxidant enzymes in neuronal cells. Therefore, these results corroborate findings of oxidative stress prevention in hippocampal neuronal cells by EAEP by regulating the expression of BDNF and antioxidant enzymes via activation of both TrkB/BDNF and Akt/Nrf2/antioxidant enzyme pathways. Mar. Drugs 2020, 18, 372 5 of 12

2.5. K252a and MK2206 Inhibit the Neuroprotective Effects of EAEP Subsequently, we found that the neuroprotective effects of EAEP affect both the BDNF/TrkB pathway and the Akt/Nrf2/antioxidant enzymes by treating with K252a, a TrkB inhibitor, or MK2206, a selective Akt inhibitor, We determined that the inhibitory effect dramatically neutralized the antioxidant and neuroprotective effects of EAEP in oxidative stress-induced HT-22 cells (Figure 5). Pre-treatment with either K252a or MK2206 in combination with EAEP significantly reduced the neuroprotective effect of EAEP against glutamate-treated HT-22 cells (Figure 5A,C). In addition, the inhibitor treatment interfered with the ability of EAEP to reverse ROS synthesis in oxidative stress- induced hippocampal neuronal cells (Figure 5B,D). Furthermore, both K252a and MK2006 reversed the antioxidant action of EAEP by reducing the expression of antioxidant enzymes including HO-1, NQO-1, and GCLC in oxidative stress-induced HT-22 cells (Figure 6). In particular, the expression of BDNF and p-TrkB was decreased when the inhibitors were pre-treated with EAEP (Figure 7). Collectively, these results suggest that the activation of Akt is closely associated with the BDNF/TrkB pathway as well as the expression of antioxidant enzymes in neuronal cells. Therefore, these results corroborate findings of oxidative stress prevention in hippocampal neuronal cells by EAEP by regulating the expression of BDNF and antioxidant enzymes via activation of both TrkB/BDNF and Mar.Akt/Nrf2/antioxidant Drugs 2020, 18, 372 enzyme pathways. 6 of 13

Figure 5. Treatment with K252a or MK-2206 inhibits neuroprotective activity of ethyl acetate extract of EnteromorphaFigure 5. Treatment prolifera (EAEP).with K252a HT-22 or MK-2206 cells were inhibits pre-incubated neuroprotective with or without activity EAEPof ethyl in acetate combination extract withof Enteromorpha K252a or MK-2206 prolifera for 30(EAEP). min before HT-22 glutamate cells were challenge. pre-incu Afterbated 12 h,with the cellor viabilitywithout andEAEP ROS in levelscombination were measured with K252a as described or MK-2206 in the for Materials 30 min and before Methods. glutamate (A,C )challenge. Cell viability; After and 12 ( Bh,,D the) ROS cell levels.viability Data and represent ROS levels the mean were measuredSEM values as baseddescribed on quintuple in the Materials determinations. and Methods. ** p < 0.01 (A versus,C) Cell ± Mar. Drugsglutamate-treatedviability; 2020, 18 and, 372 (B group;,D) ROS## p levels.< 0.01 versusData represent EAEP with the glutamate-treated mean ± SEM group.values basedabsent, on+ quintuplepresent.6 of 12 determinations. ** p < 0.01 versus glutamate-treated group; ## p < 0.01 versus EAEP− with glutamate- treated group. − absent, + present.

Figure 6. TreatmentTreatment with with K252a K252a or or MK-2206 MK-2206 inhibits inhibits neurop neuroprotectiverotective effects effects of of ethyl ethyl acetate acetate extract extract of ofEnteromorphaEnteromorpha prolifera prolifera (EAEP).(EAEP). (A (A) )Immunoblotting Immunoblotting for for protein protein expr expressionession related related with AktAkt/Nrf2/Nrf2 B C pathway,pathway, ((B)) relativerelative densitydensity ofof p-Akt,p-Akt, ((C)) NQO-1,NQO-1, GLCL,GLCL, andand HO-1.HO-1. HT-22 cellscells were pre-incubatedpre-incubated with or without EAEP in combination with K252a or MK-2206 for 30 min before glutamate challenge. with or without EAEP in combination with K252a or MK-2206 for 30 min before glutamate challenge. After 12 h, the expression of p-Akt, HO-1, NQO-1, GCLC, or β-actin was determined as described in After 12 h, the expression of p-Akt, HO-1, NQO-1, GCLC, or β-actin was determined as described in the Materials and Methods. The data were obtained from three independent experiments. ** p < 0.01 the Materials and Methods. The data were obtained from three independent experiments. ** p < 0.01 versus glutamate-treated group; ## p < 0.01 versus EAEP in the glutamate-treated group. absent, versus glutamate-treated group; ## p < 0.01 versus EAEP in the glutamate-treated group. − −absent, + + present. present.

Figure 7. Treatment with K252a or MK-2206 inhibits neuroprotective effects of ethyl acetate extract of Enteromorpha prolifera (EAEP). (A) Immunoblotting for protein expression related with BDNF/TrkB pathway, and (B) relative density of BDNF and p-TrkB. HT-22 cells were pre-incubated with or without EAEP in combination with K252a or MK-2206 for 30 min before glutamate challenge. After 12 h, the expression of BDNF, p-TrkB, or β-actin was examined as described in the Materials and Methods. The data were obtained from three independent experiments. ** p < 0.01 versus glutamate- treated group; ## p < 0.01 versus EAEP with glutamate-treated group. − is absence, + is present.

3. Discussion Enteromorpha prolifera (EP), which is a green alga, has been used as food and traditional medicine in Eastern Asia for a long time. EP exhibits anti-diabetic, antioxidant, and anti-inflammatory activities [17–20,25,26]. Recently, the antioxidant properties of ethyl acetate extract of EP (EAEP) were attributed to the presence of diverse phytochemical ingredients, such as chlorophyll, phycocyanins, flavonoids, phenolic compounds, and polyssacharides [17,19,21,23]. In addition to phytochemicals, EAEP contains carotenoid-derived compounds associated with neuroprotective effect [12–15]. However, the neuroprotective effects of EAEP against oxidative stress-induced neurodegeneration have yet to be reported.

Mar. Drugs 2020, 18, 372 6 of 12

Figure 6. Treatment with K252a or MK-2206 inhibits neuroprotective effects of ethyl acetate extract of Enteromorpha prolifera (EAEP). (A) Immunoblotting for protein expression related with Akt/Nrf2 pathway, (B) relative density of p-Akt, (C) NQO-1, GLCL, and HO-1. HT-22 cells were pre-incubated with or without EAEP in combination with K252a or MK-2206 for 30 min before glutamate challenge. After 12 h, the expression of p-Akt, HO-1, NQO-1, GCLC, or β-actin was determined as described in the Materials and Methods. The data were obtained from three independent experiments. ** p < 0.01 Mar. Drugsversus2020 glutamate-treated, 18, 372 group; ## p < 0.01 versus EAEP in the glutamate-treated group. − absent, +7 of 13 present.

Figure 7. 7. TreatmentTreatment with with K252a K252a or MK-2206 or MK-2206 inhibits inhibits neurop neuroprotectiverotective effects of eff ethylects acetate of ethyl extract acetate of extractEnteromorpha of Enteromorpha prolifera (EAEP). prolifera (A(EAEP).) Immunoblotting (A) Immunoblotting for protein expr for proteinession related expression with relatedBDNF/TrkB with BDNFpathway,/TrkB and pathway, (B) relative and (Bdensity) relative of densityBDNF and of BDNF p-TrkB. and HT-22 p-TrkB. cells HT-22 were cells pre-incubated were pre-incubated with or withwithout or without EAEP in EAEP combination in combination with K252a with or K252a MK-2206 or MK-2206 for 30 min for30 before min beforeglutamate glutamate challenge. challenge. After After12 h, 12the h ,expression the expression of BDNF, of BDNF, p-TrkB, p-TrkB, or β-actin or β-actin was wasexamined examined as described as described in the in Materials the Materials and andMethods. Methods. The data The were data obtained were obtained from three from independent three independent experiments. experiments. ** p < 0.01 **versusp < 0.01 glutamate- versus glutamate-treated group; ## p < 0.01 versus EAEP with glutamate-treated group. is absence, treated group; ## p < 0.01 versus EAEP with glutamate-treated group. − is absence, + is present.− + is present.

3. Discussion Enteromorpha prolifera (EP),(EP), whichwhich isis aa green alga,alga, has been used as food and traditional medicine in Eastern Eastern Asia Asia for for a long a long time. time. EP exhibits EP exhibits anti-diabetic, anti-diabetic, antioxidant, antioxidant, and anti-inflammatory and anti-inflammatory activities activities[17–20,25,26]. [17–20 Recently,,25,26]. Recently, the antioxidant the antioxidant properties properties of ethyl of ethyl acetate acetate extract extract of ofEP EP (EAEP) (EAEP) were attributed to the presence of diverse phytochemicalphytochemical ingredients, such as chlorophyll, phycocyanins, flavonoids,flavonoids, phenolic compounds, and polyssacharides [[17,19,21,23].17,19,21,23]. In addition to phytochemicals, EAEP containscontains carotenoid-derived carotenoid-derived compounds compounds associated associated with neuroprotectivewith neuroprotective effect [12 –effect15]. However, [12–15]. theHowever, neuroprotective the neuroprotective effects of EAEP effects against of EAEP oxidative against stress-induced oxidative stress-induced neurodegeneration neurodegeneration have yet to behave reported. yet to be reported. Recent studies suggest that the stabilization of the BDNF autocrine loop is critical for prevention and treatment of neurodegenerative diseases. Based on these findings, we hypothesized that the antioxidant property of EAEP may be related to the synthesis of both BDNF and antioxidant enzymes in oxidative stress-exposed neuronal cells [5,9,32,34]. In this study, we found that EAEP exhibits both neuroprotective and antioxidant properties mediated via BDNF expression through the ERK/CREB/TrkB pathway and antioxidant enzymes such as HO-1, NQO-1, and GCLC mediated via the Akt/Nrf2 pathway, in oxidative stress-induced neuronal cells. In contrast, EAEP attenuated the expression of JNK and p38, which belong to MAPKs along with ERK, which were reduced in oxidative stress-induced neuronal cells. Furthermore, treatment with K252a, an inhibitor of TrkB, and MK2206, a selective Akt inhibitor suppressed the neuroprotective effect of EAEP. Moreover, the expression of BDNF and antioxidant enzymes was reduced by pre-incubating the inhibitors with EAEP in oxidative stress-induced neuronal cells, which demonstrate that the activation of Akt may enhance the expression of BDNF and stabilize the activation of TrkB/Akt pathway in neuronal cells. The neuroprotective and antioxidant mechanisms of EAEP are possibly correlated with the activation of neurotrophic signaling pathways to promote neuronal survival in neurodegenerative conditions. BDNF is the primary neurotrophic factor that enhances not only cell proliferation and growth but synaptic plasticity by activating TrkB, its receptor [7,8]. In addition, treatment with p-TrkB, an activated form of TrkB, leads to the activation of Akt, extracellular signal-regulated kinase (ERK), and cAMP-response element binding protein (CREB) pathway [5,8,31]. Recently, it was reported that the downstream phosphorylation of ERK, which is a mitogen-activated protein kinase (MAPK), activated CREB transcription, regulating the expression of BDNF [35,36]. In contrast, the phosphorylation of JNK and p38, which are also MAPKs, leads to apoptosis and cell Mar. Drugs 2020, 18, 372 8 of 13 death [37,38]. Based on these results, we suggest that ERK may promote the expression of BDNF and TrkB in oxidative stress-induced HT-22 cells. In addition to neuronal defense system, the production of antioxidant enzymes was enhanced via activation of the TrkB/Akt/Nrf2 pathway in oxidative stress-induced neuronal cells. Glutamate toxicity is a major contributor to pathological cell death within the nervous system and appears to be mediated by reactive oxygen species [39]. High concentration of glutamate inhibits the uptake of N-acetyl cysteine, which reduces the intracellular glutathione levels and subsequent oxidative stress associated with cell death such as apoptosis and necrosis [30,31]. In particular, high concentrations of glutamate trigger oxidative glutamate toxicity in HT-22 cells, which lack functional ionotropic glutamate receptors [30,31,39]. During oxidative stress, ROS such as superoxide (O2 –) • and hydrogen peroxide (H2O2) are generated mainly by mitochondria and accumulated in cells [32]. Increased ROS level is accompanied by the reduction in antioxidant enzyme expression. To suppress oxidative stress, neuronal cells activate endogenous antioxidant defense system, especially Nrf2, the key switch controlling the expression of antioxidant enzymes such as HO-1, NQO-1, and GCLC. In oxidative stress-induced neurodegenerative brains, the concentration of Nrf2 located in the cytoplasm is higher than in the nucleus and does not activate the expression of antioxidant enzymes [31]. The activation of Akt is closely linked to nuclear translocation of Nrf2 in neuronal cells [32,34]. As previously mentioned, the activation of TrkB activates Akt, which leads to nuclear translocation of cytoplasmic Nrf2. Overall, maintaining the TrkB/Akt pathway is a key strategy for the prevention and treatment of neurodegenerative diseases. The strategy is supported by evidence suggesting that EAEP reduced the formation of ROS and enhanced the phosphorylation of TrkB and Akt, and the expression of nuclear Nrf2, HO-1, NQO-1, and GCLC in oxidative stress-exposed HT-22 cells. In contrast, the addition of K252a or MK2206 attenuated the neuroprotective and antioxidant properties or EAEP by suppressing the activation of the TrkB/Akt pathway. In a previous report, we identified bioactive phytochemicals in EAEP, such as carotenoid-derived dihydroactinidiolide, canthaxanthin, fucoxanthin, violaxanthin, methionine-derived dimethylsulphoniopropionate (DMSP), chlorophyl-derived pheophorbide A, chlorophyllin, flavonoids, and phenolic compounds [23]. Based on the previous report, the neuroprotective effect of EAEP may be attributed to a synergistic effect of the bioactive compounds. In summary, this study demonstrated that EAEP exhibits neuroprotective and antioxidant activities by promoting the synthesis of BDNF and antioxidant enzymes in oxidative stress-induced neuronal cells. The benefits of EAEP, which is rich in phytochemicals, are attributed to the stabilization of the activation of the BDNF/TrkB/Akt pathway. These results demonstrate a novel neuroprotective mechanism of EAEP against oxidative stress-induced neurodegeneration, suggesting potential role as a food supplement in the treatment and prevention of neurodegenerative diseases. Further animal studies are necessary to corroborate the neuroprotective effect of EAEP for potential clinical application.

4. Materials and Methods

4.1. Chemicals Dulbecco’s Modified Eagle Medium (DMEM), 1 PBS and 1 Tris-buffered saline (TBS) were × × purchased from Welgene, Inc. (Gyeongsan, Gyeongbuk, Korea). Fetal bovine serum (FBS), 0.25% trypsin-EDTA, antibiotics, and 2,7-dichlorofluorescein diacetate (DCFDA) were obtained from Invitrogen (Carlsbad, CA, USA). K252a (a TrkB inhibitor) and MK-2206 (a specific Akt inhibitor) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). EZ-Cytox cell viability assay kit was obtained from Daeil Lab (Seoul, Korea). Specific antibodies against heme-oxygenase-1(HO-1), glutamate–cysteine ligase catalytic subunit (GCLC), and NAD(P)H quinone oxidoreductase-1 (NQO-1), NF-E2-related factor-2 (Nrf2), phosphoprotein kinase B(Akt), extracellular signal-regulated kinase (ERK), phospho-cAMP response element-binding protein (CREB), phospho-tropomyosin-related kinase receptor B(TrkB), and β-actin as well as horseradish peroxidase-conjugated IgG secondary Mar. Drugs 2020, 18, 372 9 of 13 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Specific antibodies against BDNF (sc-65514) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Muse® Annexin V & Dead Cell Assay Kits were purchased from Merck Millipore, Inc. (Darmstadt, Germany). All other chemicals used in this study were analytical grade and procured from Sigma-Aldrich (St Louis, MO, USA).

4.2. Preparation of EAEP Extract EAEP (Songwonfood, Seosan, South Korea) was prepared by a previous method [23]. Briefly, lyophilized EP (200 g) was extracted with 95% ethanol in a bath sonicator for 1 day, and the mixture was filtered through Whatman filter paper (No. 2). The process was repeated three times. The whole filtrate was concentrated using a rotary evaporator (Rikakikai Co. Tokyo, Japan). The concentrate was added to ethyl acetate and distilled water (1:1, v/v), followed by separation and evaporation of ethyl acetate layer. In a previous report, we identified a lot of phytochemicals in EAEP, including dihydroactinidiolide, canthaxanthin, fucoxanthin, violaxanthin, dimethylsulphoniopropionate (DMSP), pheophorbide A, chlorophyllin, astaxanthin, apocarotenoid, apocarotenal, chlorophyllin, lutein, zeaxanthin, flavonone, trans-stilbene, 4,40-dinitrostilbene, triiodophloroglucinol, naringenin, phenylnaringenin, xanthone [23]. Finally, the dried residue of ethyl acetate extract (2.6 g) was dissolved in dimethyl sulfoxide (DMSO).

4.3. Cell Viability Assay Cell viability was assessed according to a process reported previously [9]. HT-22 cells were pre-incubated with or without EAEP (0–100 µg/mL DMSO) for 30 min prior to glutamate treatment. After 12 h, the cell viability was assessed using an EZ-Cytox cell viability assay kit according to the manufacturer’s instructions. The absorbance at 450 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The percentage of surviving cells was determined relative to the control values.

4.4. Measurement of Intracellular ROS Level

The level of intracellular ROS was measured using 20,70-dichlorofluorescein diacetate (DCFDA) following a previous method [9]. After glutamate treatment for 7 h, the cells were stained with 10 µM DCFDA in Hank’s balanced salt solution (HBSS) for 30 min in darkness. The plate was measured by microplate reader (Beckman Coulter DTX 880 Multimode Detector, Brea, CA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 525 nm.

4.5. Flow Cytometry Analysis To measure apoptotic bodies, HT-22 cells were seeded. After glutamate treatment for 12 h, all the cells were harvested. Dead cells including both apoptotic cells and necrotic cells were measured by using Muse Annexin V & Dead Cell Assay kit following the manufacturer’s instruction. Finally, stained cells were analyzed by a flow cytometer (MuseTM Cell Analyzer, Merck Millipore, Darmstadt, Germany) with Muse 1.1.2 analysis software.

4.6. Protein Determination Proteins were measured with a Bio-Rad protein assay dye reagent [40]. The cell lysate mixed with a 1:20 diluted dye reagent was incubated at room temperature for 10 min. The mixture was measured with a spectrophotometer at 595 nm. Bovine serum albumin (BSA) was used to obtain the standard curve in the range of 0.2–1.0 mg/mL.

4.7. Extraction of Nuclear and Cytosolic Protein Nuclear and cytosolic proteins were fractionated using a Nuclear Extraction Kit (Cayman Chemicals, Ann Arbor, MI) according to the manufacturer’s instructions. Briefly, cells were harvested Mar. Drugs 2020, 18, 372 10 of 13

and centrifuged (3000 rpm, 5 min) at 4 ◦C. The cell pellets were mixed with hypotonic buffer containing a phosphatase inhibitor and a protease inhibitor. After 10 min of incubation on ice, the cells were treated with 10% Nonidet P40 Assay Reagent. Nuclei were recovered by centrifugation (14,000 rpm, 30 s), and the supernatant was stored as a cytoplasmic extract at 80 C until use. The nuclei were extracted − ◦ with Nuclear Extraction Buffer for 30 min on ice. Insoluble material was removed by centrifugation (14,000 rpm, 10 min). Finally, the supernatant was used as a nuclear extract.

4.8. Western Blot Analysis Western blot analysis was used to determine the protein expression of HO-1, NQO-1, GCLC, Nrf2, p-Akt, p-TrkB, BDNF, p-ERK, p-CREB, Lamin B, and β-acitn in HT-22 cells. Briefly, cellular proteins were extracted using the PRO-PREP Protein Extraction solution (iNtRON Biotechnology, Gyeonggi-do, Korea) according to the manufacturer’s instructions. The proteins (20–60 µg) obtained from the supernatant were resolved by SDS-PAGE, and transferred onto a polyvinylidene fluoride (PVDF) membrane. The nonspecific binding of antibodies was blocked using 5% BSA in TBS buffer (20 mM Tris-HCl + 150 mM NaCl, pH 7.4) for 2 h, and the membranes were probed with different primary antibodies. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG for 1 h for the immunoblotting analysis, followed by visualization using the WEST OneTM western blot detection system (iNtRON Biotechnology, Inc, Gyeonggi-do, Korea). The relative density of the protein expression was quantitated by densitometry (Image J, National Institutes of Health, Bethesda, Maryland, USA).

4.9. Statistical Analysis All the results were expressed as the mean SEM. The statistical analysis was performed using ± SPSS 24.0 program (SPSS Inc., Chicago, IL, USA). Data were analyzed by one-way analysis of variance (ANOVA) followed by an LSD test and post-hoc comparison with Duncan’s multiple-range test. Statistical significance was considered at * p < 0.05, ** p < 0.01, or *** p < 0.001 for the comparison involving only glutamate-treated HT-22 cells. The statistical significance was considered at # p < 0.05, ## p < 0.01, or ### p < 0.001 for comparison with glutamate-treated HT-22 cells exposed to EAEP 100 µg/mL.

5. Conclusions The present study demonstrated that EAEP exerts neuroprotective action by protecting against glutamate-induced apoptosis via the TrkB/Akt signaling pathway in oxidative stress-induced hippocampal neuronal cells. Our study suggests that the neuroprotective effect of EAEP may provide further information for the application of EAEP as a candidate for the prevention and treatment of neurodegenerative disorders.

Author Contributions: Conceptualization, M.R.K. and S.Y.B.; methodology, M.R.K. and S.Y.B.; software, S.Y.B.; validation, S.Y.B.; formal analysis, S.Y.B.; investigation, S.Y.B.; data curation, M.R.K.; writing—original draft preparation, S.Y.B.; writing—review and editing, M.R.K.; visualization, S.Y.B.; supervision, M.R.K.; project administration, M.R.K.; funding acquisition, M.R.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was a part of the project titled ‘Chungcheong Sea Grant’ funded by the Ministry of Oceans and Fisheries, Korea (grant number 20170341) and supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number 2017R1D1A3B03027867). Conflicts of Interest: The authors declare no conflict of interest. Mar. Drugs 2020, 18, 372 11 of 13

Abbreviations

Akt protein kinase B BDNF brain-derived neurotrophic factor CREB cAMP response element-binding protein EAEP ethyl acetate with Enteromorpha prolifera GCLC glutamate–cysteine ligase catalytic subunit HO-1 heme oxygenase-1 NQO-1 NAD(P)H quinine oxidoreductase-1 Nrf2 NF-E2-related factor-2 ROS reactive oxygen species TrkB Tropomyosin-related kinase receptor B

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